Method N1 combines three previously separate methods (identification, assay, and content uniformity determination) into a single method using RP-HPLC-UV. It standardizes materials, instrumentation, and procedures across the previously separate methods to minimize differences at the border between methods. The method establishes parameters for the HPLC column, temperature, flow rate, injection volume, detection wavelength, mobile phases, and gradient program. This consolidated method aims to increase efficiency by performing multiple analyses in a single run while reducing variability introduced by borders between methods.
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Crossing Multiple Borders in Analytical Method Development
1. 8/2/2010
Method Transfer: Crossing Multiple Borders
Xiande (Andy) Wang, Ph.D.
Analytical Research and Development
Cordis Corporation,
A Johnson & Johnson Company,
Warren, NJ 07059
IVT Annual Method Validation 2010
Outline
1. Lifecycle of Analytical Methods
2. Border I: Between Methods
– Case study: combination of 3 methods into one
3. Border Between Instrumentation/Technique
– Case study: validation of an HPLC and UPLC method side by side
4. Border Between Groups
– Case study: Troubleshooting cross functions during method transfer
5. Conclusions
1
2. 8/2/2010
Life Cycle of an Analytical method
Method
Development
Method Method
transfer validation
Life Cycle of Analytical methods
Development
transfer validation
2
3. 8/2/2010
Life Cycle of Analytical Methods - Examples
Development
transfer validation
Life Cycle of Analytical Methods - Examples
UPLC HPLC Development
HPLC UPLC
UPLC HPLC
UPLC HPLC UPLC HPLC
HPLC UPLC HPLC UPLC
UPLC HPLC UPLC HPLC
validation
transfer
3
4. 8/2/2010
Borders to Cross in Lifecycle of Method
Border II
UPLC HPLC Development
Border I
HPLC UPLC
UPLC HPLC
Border III
UPLC HPLC UPLC HPLC
HPLC UPLC HPLC UPLC
UPLC HPLC UPLC HPLC
validation
transfer
Border I: Between Methods
UPLC HPLC
UPLC
HPLC
UPLC HPLC
4
5. 8/2/2010
Questions Around Border I
• Can the method be used interchangeably?
• Methods require the same instrumentation, column, reagents,
materials?
• Solution (standard, sample, mobile phase) storage and stability
• Timing of validation, transfer; effective date, versions
• Will methods be run in the same or different lab?
• Sample shipping, storage at different environment
• Consistency in any other areas
How is HPLC Assay Method Developed?
Method Objectives
– Stability indicating
• Peak purity
• Resolution of all species
• LC-MS compatible
• Elution of all species/compounds
– Long gradient method
– Orthogonal
– Robust
– Sensitivity
5
6. 8/2/2010
Systematic HPLC Assay Method Development
Samples are Stressed samples Representative
stressed under are analyzed with stressed samples are
different a generic method chosen
conditions
Method screening is
The method is A primary method conducted with
optimized is identified selected samples
Stressed samples are The primary method is ready
analyzed with for further optimization /
optimized method validation
11
General guidelines for HPLC Column Selection
• Select high-purity silica-based columns
• C18 and C8– Hydrophobic, retentive and stable
• Phenyl – medium polarity components; unique selectivity for
aromatics
• Hydrophilic end-capped phases (retentive for water soluble
compounds)
• Polar-embedded phases (amide, carbamate, ether, sulfonamide)
– Less tailing for basic analytes
– “Orthogonal” to C8/C18, No phase collapse
• Explore selectivity differences between C18, polar-embedded or
phenyl bonded phases
– Consult column selectivity chart
– For low pH Applications, select column resistant to hydrolytic cleavage
(e.g., StableBond, X-Bridge C18)
– For high-pH application, select columns stable at high pH (e.g., Gemini, X-
Bridge, Extend, Luna)
12
M.W. Dong, Modern HPLC for Practicing Scientists, Wiley, 2006, Chap. 3.
6
7. 8/2/2010
Some Popular HPLC Columns
• Waters: Symmetry, SunFire, XTerra *, ACQUITY*, X-Bridge*, Atlantis,
NovaPak, m-Bondapak, Spherisorb
• Agilent: Zorbax StableBond, Eclipse XDB, Extend C18*, Bonus
• Phenomenex: Luna*, Prodigy, Synergi*, Gemini*
• Supelco: Discovery, Ascentis, Supelcosil
• Varian: Inertsil, Polaris*
• Thermo: HyPURITY, Hypersil, Prism, Hypersil Gold *
• MacMod: ProntoSIL, ACE (Adv. Chrom. Tech.)
• YMC: YMCbasic, Pack Pro
• Eka Chemicals: Kromasil
• GL Sciences: Inertsil
• Macherey Nagel: Nucleosil
• Merck KGaA: Chromolith (Monolith)
• Bischoff: ProntoSIL*
• Grace: Vydac, Platinum (Alltech)
• Dionex: Acclaim, Acclaim PA, Acclaim PA2*
Columns based on high-purity silica are underlined. Hybrid particles are in bold.
Phases stable in high pH are italicized and marked with *.
13
M.W. Dong, Modern HPLC for Practicing Scientists, Wiley, 2006, Chap. 3.
HPLC Assay Method Development: Column Screening
E. F. Hewitt, P.
Lukulay, and S.
Galushko,
J Chromatography A,
1107, 79.
7
8. 8/2/2010
HPLC Column Screen Set: An Example
Orthogonal Screening – Columns
Stationary Phase Column pH Rangea Manufacturer Part Number
C18 – Twin Technology Gemini C18, 5 μm, 110A, 4.6 x 150 mm 1-12 Phenomenex 00F-4435-E0
Phenyl with Hexyl (C6) linker, Luna Phenyl-Hexyl, 3 μm, 4.6 x 150 mm 1.5-10 Phenomenex 00F-4256-E0
endcapped
C18-20% C loading Discovery HS-C18, 3μm, 4.6 x 150 mm 2-8 Supelco 569252-U
C18 – polar embedded, hybrid XTerra RP18, 3.5 μm, 4.6 x 150 mm 1-12 Waters 186000442
particle with Shield Technology
C18– silica Sunfire C18, 3.5 μm, 4.6 x 150 mm 2.8 Waters 186002554
Pentafluorophenyl Curosil PFP, 3 μm, 4.6 x 150 mm 2-7.5 Phenomenex 00F-4122-E0
a
Columns were screened only against mobiles phases within their compatible pH range.
Slide courtesy of H. Rasmussen et al
15
HPLC Screening Conditions: An Example
Orthogonal Screening Method Description
a
Time (min) %Water %Acetonitrile % Modifier Flow Rate (ml/min)
0 85 10 5 1.0
40 10 85 5 1.0
45 10 85 5 1.0
45.10 85 10 5 1.0
60 85 10 5 1.0
Injection Volume 5 μL
Detection 280 nm; DAD (190 – 400 nm)
Column Temperature Ambient
o
Sample Temperature 5C
aModifierstock solutions are prepared at a concentration 20 times higher than the desired mobile phase concentration since mobile phases are prepared at time of use
with the HPLC quaternary pump.
Modifier Mobile Phase Approximate pH
Concentration
Trifluoroacetic Acid (TFA) 0.05% 2
Formic Acid 0.1% 2.8
Ammonium Acetate + Acetic Acid 8 mM + 0.1% 4
Ammonium Acetate 8 mM 7
Ammonium Acetate + Ammonium 8 mM + 0.05% 10.2
Hydroxide
Ammonium Hydroxide 0.05% 10.8
16
Slide courtesy of H. Rasmussen et al
8
9. 8/2/2010
Summary of HPLC Assay Method Development
• There is a systematic approach for assay method
development.
• Column screening is an effective tool.
• It will enhance efficiency by narrowing down the list of
columns for screening/selection.
• It is critical to seek input from other labs (QA, QC) during
method development.
• It’s important to look at the big picture across methods.
What is Important in Developing HPLC CU Method?
Method Objectives
– Short
– Specific
– Isocratic preferred
– Robust
– Compatible with assay method
– Can be used for dissolution testing
9
10. 8/2/2010
Systematic HPLC CU Method Development
samples are Specificity, retention, No
analyzed with a peak shape etc.
generic method acceptable
Yes
The method is A primary method is Method screening is
optimized identified conducted
The primary method is ready for
further optimization / validation
19
HPLC CU Method Development: Column Screening
E. F. Hewitt, P.
Lukulay, and S.
Galushko,
J Chromatography A,
1107, 79.
10
11. 8/2/2010
What is Important in Developing HPLC Method for
Dissolution Testing?
– Short
– Specific
– Isocratic preferred
– Robust
– Compatible with CU method
– Sensitive
How to Minimize Border I (Between Methods)
• Use same materials/chemicals: grade, vendor
• Adopt similar standard/sample solution: procedure of preparation,
concentration, pH, storage, expiry
• Use same column: vendor, stationary phase, dimension, particle size;
alternative column
• Use same Instrumentation
• Be consistent in write-up: same product description, formulation
number, etc.
11
12. 8/2/2010
Case Study: Combination of 3 methods into 1
Method Method title
number
N1 Identification, Assay and Content Uniformity Determination of … by
RP-HPLC-UV
O1 Identification and Assay of … by RP-HPLC-UV
O2 Determination of Content Uniformity of … by RP-HPLC-UV
O3 Identification of … by UV Spectroscopy
Method O1: Identification and Assay of … by RP-HPLC-UV
Parameter Value
HPLC Column Agilent, Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm
x 3.5 µm 02
.00
Flow Rate 1.0 ± 0.1 mL/min
Injection Volume 25 µL
Column 01
.05
40 ± 2 °C
Temperature
Detection 278 nm.
Note: If using an Agilent DAD detector, set the bandwidth to 4 01
.00
Wavelength
AU
nm and the reference wavelength off.
A: 80:20% (v/v) 0.02% formic acid:THF
Mobile Phases B: 75:20:5:0.02% (v/v/v/v), acetonitrile: THF : water:
00
.05
formic acid
Time
% Mobile Phase A % Mobile Phase B
(minutes)
00
.00
Gradient 0.0 55 45
Program 3 55 45 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 .0 1.0 1.0 1 .0 1 .0 1.0
.0 .0 .0 .0 .0 .0 .0 .0 .0 0 0 1 0 2 0 3 0 4 0 5 0
(Linear Gradient
7 42 58 Mu s
in te
Profile)
13 10 90
15 55 45
12
13. 8/2/2010
Method O2: Determination of Content Uniformity of
and Dissolution … by RP-HPLC-UV
Parameter Value
Phenomenex Luna C18 (2), 4.6
x 50 mm, 3 μm or Phenomenex
Column
Gemini C18, 4.6 x 50 mm, 3 μm
HPLC Column
Column 35oC ± 2oC
Temperature
Ambient (20 to 25 °C if
Autosampler
temperature control is
Temperature
available)
55:45, 0.02% v/v Formic
Mobile Phase
Acid:THF
Flow Rate 1.2 mL/minute
278 nm
Note: If using Agilent PDA
Detector
detector, set bandwidth to 4 nm
and reference wavelength off.
Injection Volume 25 μL
Run Time 10 minutes
Method O3: Identification of … by UV Spectroscopy
13
14. 8/2/2010
Method N1: Identification, Assay and Content
Uniformity Determination of … by RP-HPLC-UV
Parameter Value
R A P A M Y C IN - 4 .05 5
0.028
HPLC Column Agilent Zorbax Eclipse XDB-C18, 100 mm x 4.6 0.026
mm, 3.5 µm 0.024
0.022
Flow Rate 1.0 mL/min 0.020
3.3 90
Injection Volume 20 µL 0.018
B H T - 7 .19 6
0.016
Column o
45 C ± 2°C 0.014
Temperature 0.012
AU
0.010
Assay Identification
3.5 95
0.008
2.1 81
3.0 59
Detection 278 nm
2.5 77
0.006
4.9 79
200 to 400
Wavelength
0.004
Note: If using an Agilent DAD
detector, set the bandwidth to 4 nm and
nm 0.002
0.000
the reference wavelength off. -0.002
A: (20:80)THF: Formate Buffer -0.004
Mobile Phases
B: (75:20:5)ACN:THF:Formate Buffer 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Minutes
% Mobile % Mobile Phase
Time (min)
Phase A B 279.0
0.50
0.0 47 53 0.45
Gradient 3.0 47 53 0.40 269.0 291.0
Program 5.0 20 80 0.35
(Linear
6.0 2 98 0.30
Gradient)
AU
0.25
7.5 2 98
0.20
7.6 47 53 0.15
208.0
10 47 53 0.10
0.05
349.0
0.00
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00
nm
Benefits of a Combined Method
Benefit Details
Shortened One, instead of three, set of test methods, validation
project timelines protocols, validation reports
Enhanced Run time cut from 18 to 10min;
throughput one instrument/set-up for both assay/CU;
eliminate requirement of a UV spectrometer.
Reduce cost One , instead of two, set of HPLC column is needed
Less reference standard, organic solvent
Less sample shipment cost
Eliminated When two methods are used, possible bias could come
causes for bias from different instrument set up, different way of peak
between assay integration, different separation capacity, etc. One method
and CU eliminate all these possible causes.
14
15. 8/2/2010
Benefits of Elimination of Border I
• Same columns
• Same reagents, vendor, expiry dates
• Same materials such as glassware, HPLC vials, etc.
Assay/CU/dissolution
• No discrepancy (bias) of results due to
integration, separation capacity, etc.
• Same solution (standard, sample, mobile phase)
storage and stability
• Transfer at the same time
• Same method, same version Assay/CU/dissolution Assay/CU/dissolution
• One HPLC system needed
• Minimize sample shipping, storage
at different environment
Summary of Strategy to Minimize/Eliminate Border I
• Assay, CU, and dissolution (HPLC) methods have different objectives and hence
the strategy for method development may be different.
• However, it is important to keep the other method in mind during method
development.
• Column screening is an effective tool for assay, CU or dissolution methods.
• It is effective to narrow down the list of columns for screening/selection.
• It is critical to seek input from other labs (QA, QC) during method development.
• It’s important to leverage the knowledge, as much as possible, of one method
and apply it to another.
• It offers a lot of benefit to use similar or the same procedures, instrumentation
and materials for all methods, and transfer them at the same time.
15
16. 8/2/2010
Border II: HPLC vs. UPLC
UPLC HPLC
UPLC
HPLC
UPLC HPLC
UPLC vs. HPLC: Example of Assay Method
Ref: L. Pereira, Poster at Pittcon 2007, Chicago, Illinois, February 2007.
16
17. 8/2/2010
UPLC vs. HPLC: Example of CU Method
http://www.waters.co
m/waters
Issues Around Border II (HPLC vs. UPLC)
• Availability of instruments at all sites
• Different UHPLC systems have different design, not a direct
transfer
• Training/knowledge and technical challenges on the new
instruments
• Limited availability and technical challenges of columns
• Project history
• Cost
17
18. 8/2/2010
How to Minimize Border II (HPLV vs. UPLC)
• Use the same stationary phase with different dimension
and particle size
• Same mobile phase but slight different gradient
• Same protocol template
• Use the same solutions and validate the methods side by
side
Case study: Validation of HPLC and UPLC Assay Method Side by Side
Chromatograms of the Final Methods
RAPAMYCIN - 4.055
0.028
0.026
0.024
0.022
0.020 HPLC
3.390
0.018
BHT - 7.196
0.016
0.014
AU
0.012
0.010
UPLC
3.595
0.008
2.181
3.059
2.577
4.979
0.006
0.004
0.002
0.000
-0.002
-0.004
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Minutes
RAPAMYCIN - 2.358
0.012
BHT - 4.202
0.010
0.008
0.006
AU
0.004
0.002
0.000
-0.002
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
18
19. 8/2/2010
Case study: Validation of HPLC and UPLC Assay Method Side by Side
Final Method Parameters
HPLC UPLC
Parameter Value Parameter Value
UPLC AgilentZorbax Eclipse XDB-C18, 100
HPLC Column Agilent Zorbax Eclipse XDB-C18, 100 mm x 4.6 Column mm x 3.0 mm, 1.8 µm
mm, 3.5 µm Flow Rate 0.7 mL/min
Flow Rate 1.0 mL/min Injection
5 µL
Injection Volume 20 µL Volume
Autosampler 10 oC
Column o
45 C ± 2°C Temperature
Temperature Column
45 oC ± 2 °C
Assay Identification Temperature
Sampling
Detection 278 nm Assay
200 to 400 Rate
Wavelength Note: If using an Agilent DAD 278 nm
detector, set the bandwidth to 4 nm and
nm
Detection Note: If using an Agilent
the reference wavelength off. Wavelength DAD detector, set the 20
A: (20:80)THF: Formate Buffer bandwidth to 4 nm and (points/sec)
Mobile Phases
B: (75:20:5)ACN:THF:Formate Buffer the reference wavelength
off.
% Mobile % Mobile Phase A: (20:80)THF: Formate Buffer
Time (min) Mobile
Phase A B B: (75:20:5)ACN:THF:Formate
Phases
0.0 47 53 Buffer
% Mobile % Mobile
Gradient 3.0 47 53 Time (min)
Phase A Phase B
Program 5.0 20 80 0.0 47 53
Gradient
(Linear 2.0 47 53
6.0 2 98 Program
3.5 20 80
Gradient) (Linear
7.5 2 98 Gradient)
4.2 2 98
5.2 2 98
7.6 47 53 5.3 47 53
10 47 53 6.5 47 53
6.5 minutes (Retention time is ~2.5 min
Run Time
for sirolimus, ~4.3 min for BHT.
Seal Wash Acetonitrile
Acetonitrile/water: 50/50, 600 µL
Weak Wash
volume
Case study: Validation of HPLC and UPLC Assay Method Side by Side
Accuracy (spiked recovery)
Actual
Individual % Mean % %
~Level Concentration
Recovery Recovery RSD
(μg/mL) HPLC
99.9
40% 67.4 99.7 99.9 0.1
99.9
99.5
UPLC
100% 151.7 99.8 99.6 0.2
99.5
100.6
160% 242.7 100.6 100.5 0.2
100.3
Actual
Individual % Mean % %
~Level Concentration
Recovery Recovery RSD
(μg/mL)
100.9
40% 67.4 101.0 101.0 0.1
101.1
99.4
100% 151.7 99.6 99.6 0.2
99.8
99.3
160% 242.7 98.9 99.0 0.2
98.9
19
22. 8/2/2010
Validation of HPLC and UPLC CU Method Side by Side:
Linearity
HPLC
UPLC
R=0.9997
R=1.0000
Validation of HPLC and UPLC CU Method Side by Side:
Method Equivalency
Determi- %LC %LC with %LC old
%LC with Determi-
nation with old new CU
new nation
CU method method
method
method 1 98.4 98.7
1 102.0 101.3 2 99.2 99.6
2 101.6 100.6 3 98.7 99.9
3 101.3 100.8 4 100.1 100.3
4 100.4 100.4 5 100.0 99.9
5 102.6 102.2 6 100.4 100.6
6 101.0 100.7 7 100.4 100.2
7 101.7 101.1 8 100.1 100.5
8 100.8 100.6 9 100.7 100.2
9 101.6 101.4 10 99.9 100.4
10 101.4 101.0 Mean
99.8 100.0
Mean 101.4 101.0 (n=10)
(n=10) %RSD
0.8 0.6
%RSD 0.6 0.5 (n=10)
Absolute Absolute
difference 0.4 Difference 0.2
(%) (%)
HPLC UPLC
22
23. 8/2/2010
Summary of Case Study to Minimize Border II (HPLC vs. UPLC)
• Use the same stationary phase with different dimension and
particle size
• Same mobile phase but slight different gradient
• Same protocol template
• Use the same solutions and validate the method side by side
• The knowledge of both methods were exchanged during the
process
• HPLC and UPLC method proved to be equivalent and can be used
interchangeably
Border III. Between Functions
Border II
UPLC HPLC Development
Border I
HPLC UPLC
UPLC HPLC
Border III
UPLC HPLC UPLC HPLC
HPLC UPLC HPLC UPLC
UPLC HPLC UPLC HPLC
validation
transfer
23
24. 8/2/2010
III-a: Method Development vs. Validation
• Often reside in the same functional group
• Natural redundancy in terms of robustness, forced
degradation, alternative columns
Method
Development
Method Method
transfer validation
What Is Analytical Method Transfer
• Protocol driven study with pre-defined acceptance criteria
• Transfer of validated analytical procedures to a new
laboratory
• Verification of a method’s suitability for its intended use
• Demonstration of a laboratory’s proficiency in running a
particular method
• No official guidelines
24
25. 8/2/2010
Options for Method Transfer
• Comparative testing:
A set of samples are tested in both labs and resulting data are compared with
predetermined acceptance criteria.
• Co-validation between two labs:
The receiving laboratory is involved in method validation but have to identify
which validation parameters are to be generated or challenged by the two
labs.
• Complete or partial method validation:
A repeat of method validation either completely or partially.
• Transfer waiver (omission of formal validation):
Needs justification as to why method transfer was not needed. For example,
lab is already testing the product.
Border III-b: Method Transfer and Validation
• Method transfers are closely related to
validation
• Method transfer is more challenging because
multiple laboratories and companies are
involved
– Different approaches to Validation and Transfer
– Different expectations of what is an acceptable
validation
– Different instruments and facilities
25
26. 8/2/2010
Method Transfer and Validation
Method transfer Method validation
• Can be part of the validation • Protocol driven study with pre-defined
acceptance criteria
• Protocol driven study with pre-defined
acceptance criteria • Validation of analytical procedures in a
laboratory
• Transfer of validated analytical procedures
to a new laboratory • Verification of a method’s suitability for its
intended use
• Verification of a method’s suitability for its
intended use in a new laboratory • Demonstration of a laboratory’s
proficiency in running a particular method
• Demonstration of a laboratory’s
proficiency in running a particular method • http://www.ich.org/LOB/media/MEDIA41
7.pdf;
• No official guidelines http://www.fda.gov/cder/guidance/2396d
ft.pdf
Objectives of Method Transfer
• Maintain the validated state of the method and meet all
regulatory requirements
• Minimize surprises!
– Open and responsive communication
– Pre-determined expectations
– Clearly documented and communicated technical details
– Pre-transfer evaluation by experienced technical staff at
receiving site
– Technical contact available for troubleshooting at
transferring site
26
27. 8/2/2010
Typical Method Transfer Steps
• Discussions Initiated
• Review of Method and Validation
• Laboratory Evaluation
• Protocol (Transfer or validation) Written
• Protocol Approved
• Experimental evidence from a transfer study generated
• Report (Transfer or validation) Written
• Report Approved
• Transfer Complete
Preparation for a Method Transfer
• Method
– Details about method
– Specific instrument
• Method development history report
• Training/discussion on the method
• Materials
– Reference Standard
– Samples for Evaluation
– Difficult to purchase supplies
• Specifications
• Technical Contact
• Details about product
27
28. 8/2/2010
Prior to Formal Method Transfer
• Receiving laboratory should perform the
method
– Helps to determine where there are differences
and gaps in documentation
• Lack of detailed test method instructions
– Assay Conditions
– Calculations
– System Suitability
– Differences with instrumentation or reagents
Prior to Formal Method Transfer
• Training of Personnel
– Review of relevant SOPs
– Observation of test procedure
– Performing test procedure
• Helpful to include development, qualification
and validation reports to recipient laboratory
28
29. 8/2/2010
Method Validation and Transfer
• Method transfers are closely related to validation
• Method transfer is usually part of validation
Method
Development
Method Method
transfer validation
Border III-c: Method Transfer and Method Development:
Before or During Method Development
• Define goals of end method
• Dynamic platform of communication
• Exchange of knowledge
29
30. 8/2/2010
Border III-c: Method Transfer and Development:
After method Transfer
• Is the method inadequate by today’s scientific standard or
regulatory requirement?
• Is sufficient data available to permit simplification of the
method?
• Does monitoring of laboratory deviation suggest a need for
method improvement ?
• Do newer method for similar products significantly
outperform?
• Is the volume of testing justify further method optimization
or automation?
Border III-c: Method Transfer and Development:
During Method Transfer
– Concerns?
– Observations?
– Investigations/troubleshooting: Must involve multiple labs
30
31. 8/2/2010
Case Study: Extraneous peaks associated with HPLC vials
RAPAMYCIN - 4.133
0.010
0.009
Deactivated
glass vials
0.008
0.007
Polypro
0.006
pylene
AU
0.005
0.004
3.670
0.003
0.002
0.001 3.108
0.000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Minutes
Case Study: Extraneous peaks associated with Glass Pipettes
Rapamycin - 3.947
0.011
0.010
0.009
0.008
0.007
Unknown4 - 3.496
0.006
Unknown3 - 2.977
AU
0.005
Unknown1 - 1.690
Unknown2 - 2.526
0.004
0.003
0.002
0.001
0.000
-0.001
-0.002
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Minutes
Above: chromatograms of two different lots of glass pipettes .
Plastic transfer pipettes, or no pipettes, were recommended.
31
32. 8/2/2010
Case Study: Extraneous peaks associated with Extraction Vials
Volu
Number Price Price
Extraction Vial me Comments
per case ( $/case) ($/vial)
(mL)
10 10 224.70 22.47 Re usable. VWR Cat# 2100
Nalgene-Teflon
30 10 333.30 33.33 Re usable. VWR Cat# 2100
BD Falcon-Poly 5 500 160.00 0.32 VWR Cat # 60819-706
Propylene,
snap cap 14 500 195.30 0.39 VWR Cat # 60819-740
BD Falcon-Poly
Propylene, 15 500 220.89 0.44 VWR Cat# 21008-918
screw cap
only 2 sizes available VWR
Starplex- 5 1500 186.18 0.12
14216-262
Polypropylene
only 2 sizes available VWR
screw cap 10 1000 135.96 0.14
14216-266
NUNC Poly 15 500 192.00 0.38 Sigma Aldrich Cat# Z7204
propylene snap cap
Pre-cleaned* Sigma Aldrich
7 100 92.50 0.93
27341
Supelco pre-cleaned Pre-cleaned* Sigma Aldrich
15 100 102.50 1.03
clear glass 27342
Pre-cleaned* Sigma Aldrich
22 100 116.00 1.16
27343
Larger sizes are available u
Supelco Silanized
4 1000 267.00 0.27 request, caps not included.
Clear Glass
Sigma Aldrich Cat# 27114
Currently being used VWR
8 144 152.49 1.06
66009-984
Kimble-glass
Currently being used VWR
16 144 193.44 1.34
66009-986
Treatment to Glass Extraction Vials
• Rinse with 0.02% formic acid in acetonitrile
• Rinse with acetonitrile
• Wash with a regular washing cycle for other glassware
• Soak with 0.02% acid (formic, acetic or nitric) in water
and rinse with DI water
• Soak/sonicate in DI water
• Details are critical to ensure
accurate comparison cross labs.
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33. 8/2/2010
Standard Solution in Unwashed Glass Vials
3.50
3.00
2.50
Total Impurity Area%
2.00
1.50
1.00
0.50
0.00
0 1 2 3 4 5 6 7 8
Day
Standard Solution in Pre-cleaned Glass Vials
5
4.5
4
3.5 2ml
Total Impurity Area %
3ml
3
4ml
2.5 5ml
6ml
2
7ml
1.5 9ml
1 10ml
0.5
0
0 1 2 3 4 5 6 7 8
Day
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34. 8/2/2010
Standard Solution in Silanized Glass Vials
Standard Solutions in Glass Vials Soaked and
Sonicated in DI Water
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35. 8/2/2010
Standard Solution in Glass Vials Rinsed with MeCN 3 Times
3
2.5
2
Total Impurity area %
Direct pour
8ml- std soln-1
1.5
8ml- std soln-2
8ml- std soln-3
1 8ml- std soln-4
8ml- std soln-5
0.5
0
0 1 2 3 4 5 6 7 8
Day
Standard Solution in Glass Vials Rinsed with 0.02%
Formic Acid in MeCN 3 Times
1
0.9
0.8
0.7
Total Impurity Area%
0.6
Direct pour
8ml- std soln-1
0.5
8ml- std soln-2
0.4 8ml- std soln-3
8ml- std soln-4
0.3
8ml- std soln-5
0.2
0.1
0
0 1 2 3 4 5 6 7 8
Day
35
36. 8/2/2010
Standard Solutions in Glass Vials Treated with 0.02% Acid
(Nitric, Acetic or formic)
Standard Solution in Washed Glass Vials
Standard in Washed Extraction Vial
0.1
2
0.08
Total impurity %
3
0.06 4
0.04 5
0.02 6
0 7
0 1 2 3 4 5 6 7 9
Day 10
36
37. 8/2/2010
Standard Solution in BD Falcon Polypropylene Vials
0.16
small-1
0.14
Total Impurity area%
0.12 small-2
0.10 small-3
0.08 small-4
0.06
small-5
0.04
small-6
0.02
0.00 small-7
0 1 2 3 4 5 6 7 small-8
Day small-9
Summary to Case Study: Extraneous peaks Associate with Glassware
• Pay attention to the glass grade, vendor and treatment.
• Glass vials vary within the same lot/box.
• Rinse with 0.02% acid (formic, acetic, nitric) acid in water is effective for this
method.
• Wash the glass vials with acidic detergent is effective.
• Use of polypropylene vials eliminates the problem.
• It is critical for multiple labs to be involved, to carry our experiments and
share data with details.
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38. 8/2/2010
Method transfer vs. method development
• Start from the beginning of the lifecycle (define
goals, communicate limitations)
• Maintain a dynamic and continuous process
• Build strong partnership and co-ownership
through regular meetings, visits, design and
execute experiments together
• Transfer knowledge, not just method
Method Transfer vs. validation Vs. Development
• Dynamic platform of
communication
• Design and execute
experiments (AMERT,
Investigations)
• Share ownership Method
Development
• Exchange of knowledge
Method Method
transfer validation
38
39. 8/2/2010
Conclusions
• Analytical method lifecycle is a dynamic and continuous
process
• Transfer of knowledge, instead of method, is desired in
every stage of the life cycle
• Scientists/managers need to zoom in and zoom out to
consider needs of other projects, methods or labs.
• It enhances efficiency to eliminate borders between
methods, techniques and functional groups, as much as
possible
• Strong partnership and co-ownership is the key to
successful methods
39